1. Technical Field
The present invention generally relates to methods and compositions for enhancing expression of foreign genes in transgenic plants and other eukaryotes. More particularly, the invention relates to such methods and compositions employing Tombusvirus-based p19 gene mutants.
2. Description of Related Art
A frequently encountered obstacle in plant biotechnology efforts to express foreign genes in transgenic plants, is the induction of plant defense mechanisms against such foreign introductions, and thus the expression levels of the foreign genes are often sub-optimal. It is known that plants activate a process that is termed RNA silencing or RNA interference (RNAi) which specifically degrades the transgenically expressed foreign mRNA1. This has the direct effect of reducing or abolishing foreign protein accumulation. To counteract the effects of RNA silencing, viruses encode suppressors and the p19 protein (P19) of Tomato bushy stunt virus (TBSV) (type member of the Tombusvirus genus in the Tombusviridae) represents one of the best biochemically characterized suppressors2. P19 is a very potent suppressor and its transient presence greatly enhances the expression levels of foreign proteins in plants11-13.
Tomato bushy stunt virus (TBSV) is an RNA plant virus that infects ca. 100 plant species in more than 20 different families. TBSV particles encapsidate a single copy of a positive-sense single-stranded RNA genome of 4775 nucleotides (nt) and its spread in plants is actively regulated by the P22 cell-to-cell movement protein and P19. The nucleotide sequence of wild-type TBSV p19 is SEQ. ID NO: 1. The amino acid sequence of wild-type TBSV P19 is SEQ. ID NO: 2. FIG. 1 shows the organization of the ˜4.8-kb, single-stranded (ss) positive-sense genomic RNA (gRNA) of TBSV. The nomenclature of the five open reading frames (ORFs) (boxed regions) derives from the predicted molecular weight (in kDA) of the products. It has been shown that P19 is an intriguing multifunctional pathogenicity protein because it is a very critical host-range determinant, it is an important contributor to viral symptoms, and it has host-dependent effects on virus invasion7-10, 14. Recent studies suggest that these biological effects may be related to the activity of P19 as a suppressor of post-transcriptional gene silencing and virus-induced gene silencing11,12. It has been shown that transient co-expression of P19 prevents the onset of silencing of simultaneously introduced foreign genes, resulting in greatly (˜50-fold) enhanced transient (i.e., not transgenic) expression levels of the target foreign gene13. While this might suggest that wild-type P19 (wtP19) could possibly be developed as a tool in plant biotechnology to substantially boost levels of target gene expression in transgenic plants, the idea is tempered by the fact that P19 expression is highly toxic to many plants7,9. As a consequence, hopes for establishing transgenic plants stably expressing P19 have been essentially dashed.
Several plant viruses are known to suppress gene silencing,3 and these could also potentially serve to enhance gene expression of co-expressed value-added genes in transgenic plants. However, P19 is biochemically the best characterized and its structure has been resolved4,5. Genetic studies with site-specific P19 mutants targeting charged amino acids showed that a central region between amino acid positions 71 and 85 (especially amino acid 72) was essential for all the biological activities of P19 whereas other amino acids had host-dependent and activity-dependent effects7,11. A battery of p19 mutants were screened and a variant was identified with a single amino acid substitution at position 43 that is still active for suppression and siRNA appropriation yet is no longer toxic to plants7,11. More specifically, the arginine at position 43 of the p19 gene is replaced by a tryptophan, and the resulting variant or mutant p19 gene is referred to herein as p19/R43W or sometimes simply p19/43. The nucleotide sequence of p19/R43W is SEQ. ID NO: 3. The corresponding mutant protein is referred to herein as P19/R43W or P19/43, which has the amino acid sequence of SEQ. ID NO: 4. Certain biological properties of P19/R43W have also been described.7,10,11 During infection of a plant by TBSV expressing p19/R43W, the pathogenic properties are intermediate between those of plants infected with TBSV expressing wild-type p19 and those of plants that do not express p19. As in the case of wild-type P19 expression to suppress RNA silencing of foreign gene expression13, it was heretofore generally thought that only the relatively short term transient expression of P19/R43W in plant parts (e.g., leaves) would be possible. It was thought that the P19/R43W-expressing plant parts might provide, at best, an intermediate level of expression of a foreign gene (compared to the use of wild-type P19). Such a limited capability for enhancing transient expression (i.e., at the site of introduction in the plant part) of foreign genes would not be practical or commercially attractive for most applications. There remains a need for a practical way to improve the expression of foreign value-added genes in plants and other eukaryotic systems.